Two-step partial nitritation/Anammox process in granulation reactors: Start-up operation and microbial characterization.

A two-stage Partial Nitritation (PN)/Anammox process was carried out at lab-scale conditions to treat reject water from a municipal WWTP. PN was achieved in a granular SBR obtaining an effluent with a NH4(+)-N/NO2(-)-N molar ratio around 1.0. The microbial characterization of this reactor revealed a predominance of Betaproteobacteria, with a member of Nitrosomonas as the main autotrophic ammonium oxidizing bacterium (AOB). Nitrite oxidizing bacteria (NOB) were under the detection limit of 16S rRNA gene pyrosequencing, indicating their effective inhibition. The effluent of the PN reactor was fed to an Anammox SBR where stable operation was achieved with a NH4(+)-N:NO2(-)-N:NO3(-)-N stoichiometry of 1:1.25:0.14. The deviation to the theoretical stoichiometry could be attributed to the presence of heterotrophic biomass in the Anammox reactor (mainly members of Chlorobi and Chloroflexi). Planctomycetes accounted for 7% of the global community, being members of Brocadia (1.4% of the total abundance) the main anaerobic ammonium oxidizer detected.

Effect of interface fertilization on biodegradation of polycyclic aromatic hydrocarbons present in nonaqueous-phase liquids.

The main goal of this study was to use an oleophilic biostimulant (S-200) to target possible nutritional limitations for biodegradation of polycyclic aromatic hydrocarbons (PAHs) at the interface between nonaqueous-phase liquids (NAPLs) and the water phase. Biodegradation of PAHs present in fuel-containing NAPLs was slow and followed zero-order kinetics, indicating bioavailability restrictions. The biostimulant enhanced the biodegradation, producing logistic (S-shaped) kinetics and 10-fold increases in the rate of mineralization of phenanthrene, fluoranthene, and pyrene. Chemical analysis of residual fuel oil also evidenced an enhanced biodegradation of the alkyl-PAHs and n-alkanes. The enhancement was not the result of an increase in the rate of partitioning of PAHs into the aqueous phase, nor was it caused by the compensation of any nutritional deficiency in the medium. We suggest that biodegradation of PAH by bacteria attached to NAPLs can be limited by nutrient availability due to the simultaneous consumption of NAPL components, but this limitation can be overcome by interface fertilization.

Evaluation of biodiesel as bioremediation agent for the treatment of the shore affected by the heavy oil spill of the Prestige.

The efficiency of different bioremediation products (nutrients, microorganisms and biodiesel) was tested using tiles located in both the supra-littoral and intertidal zones of a beach that was affected by the heavy oil spill of the Prestige. Neither nutrients nor microorganisms meant an improvement with respect to the natural processes. The addition of biodiesel improved the appearance of the treated tiles and apparently accelerated the degradation of the aliphatic and aromatic fractions of the residual fuel oil. Nevertheless, PAHs degradation was similar and very high in all the treatments (80-85% after 60 days). On the other hand, the evolution with time of the amount of vanadium was similar to that of 17alpha(H),21beta(H)-hopane, so it was concluded that vanadium could also be used to estimate the extent of oil degradation in the field. These results also suggested that the residual fuel oil mineralization was very low throughout 1 year in all the treatments. Moreover, the increase of the oxygen content of the residual oil from around 1% till 4-8% indicated that the partial oxidation of hydrocarbons took place, and that the hydrocarbon oxidation products accumulated in the polar fractions. In general, the results pointed out that bioremediation techniques were not suitable for the recovery of shores affected by heavy oil spills.

Trials of bioremediation on a beach affected by the heavy oil spill of the Prestige.

The objective of this study was to assess the efficiency of several bioremediation products in accelerating the in situ biodegradation of the heavy fuel oil spill of the Prestige. Trials of bioremediation were conducted in sand, rocks and granite tiles on the beach of Sorrizo (A Coruña, NW Spain) that was polluted by the spill. Neither the added microorganisms nor the nutrients significantly enhanced the degradation rate of the fuel oil in rocks, granite tiles or sand. PAH degradation up to 80% was determined in sand and tiles. In tiles the oxygen content of the residual oil increased from 1.6% up to 8% in 90 days, which could be explained by the accumulation of products coming from the partial oxidation of the hydrocarbons. Eighteen months after the spill, the rocks of the beach were still coated by a black layer of weathered fuel oil. For this reason an oleophilic product, sunflower biodiesel was tested on a rock. The application of biodiesel accelerated the gradually clean-up of the polluted surface and could also accelerate the degradation of the residual oil.

Fossil fuel biodegradation: laboratory studies.

Biodegradation of the polycyclic aromatic hydrocarbons of creosote by undefined bacterial cultures was shown to be accompanied by the accumulation of neutral and acidic oxidation products. Formation of a number of identified neutral products is accounted for by demonstration of anomalous actions of an arene dioxygenase on the benzylic methylene and methylene carbons of napthenoaromatic hydrocarbons. Both neutral and acidic water-soluble fractions are also formed when various mixed bacterial cultures degrade weathered crude oil. While constituents of these fractions are not yet identified, the neutral materials have been shown to be toxic to developing embryos of invertebrates. These observations are discussed in relation to chemical and toxicological assessments of biodegradation of the complex chemical mixtures of fossil fuels.

Phenol removal from hypersaline wastewaters in a Membrane Biological Reactor (MBR): operation and microbiological characterisation.

In this study, two Membrane Biological Reactors (MBR) with submerged flat membranes, one at lab-scale conditions and the other at pilot-plant conditions, were operated at environmental temperature to treat an industrial wastewater characterised by low phenol concentrations (8-16 mg L(-1)) and high salinity (∼ 150-160 mS cm(-1)). During the operation of both reactors, the phenol loading rate was progressively increased and less than 1mg phenol L(-1) was detected even at very low HRTs (0.5-0.7 days). Membrane fouling was minimized by the cross flow aeration rate inside the MBRs and by intermittent permeation. Microbial community analysis of both reactors revealed that members of the genera Halomonas and Marinobacter (gammaproteobacteria) were major components. Growth-linked phenol degradation by pure cultures of Marinobacter isolates demonstrated that this bacterium played a major role in the removal of phenol from the bioreactors.

Initial Steps in the Degradation of Methoxychlor by the White Rot Fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium mineralized [ring-(sup14)C]methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] and metabolized it to a variety of products. The three most prominent of these were identified as the 1-dechloro derivative 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane, the 2-hydroxy derivative 2,2,2-trichloro-1,1-bis(4-methoxyphenyl)ethanol, and the 1-dechloro-2-hydroxy derivative 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethanol by comparison of the derivatives with authentic standards in chromatographic and mass spectrometric experiments. In addition, the 1-dechloro-2-hydroxy derivative was identified from its (sup1)H nuclear magnetic resonance spectrum. The 1-dechloro and 2-hydroxy derivatives were both converted to the 1-dechloro-2-hydroxy derivative by the fungus; i.e., there was no requirement that dechlorination precede hydroxylation or vice versa. All three metabolites were mineralized and are therefore likely intermediates in the degradation of methoxychlor by P. chrysosporium.

Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities.

Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [PAHs]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% (F1AA), 48% (TD) and 34% (AM), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n-alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% (AM), 11% (TD) and 7% (F1AA). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500-fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability.

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.

Characterization of genotoxic components in sediments by mass spectrometric techniques combined with Salmonella/microsome test.

The application of Salmonella/microsomal mammalian tests to column chromatography fractions isolated from river and marine sediments collected in the vicinity of Barcelona city, Spain, demonstrated a positive response (TA98 + S9 mix) among the polar fractions. Chemical analysis by high resolution gas chromatography coupled to negative ion chemical ionization mass spectrometry (HRGC-NICI MS) provided sensitivity and selectivity to detect several mutagenic chemical classes. Among them, nitrated PAHs, azaarenes, aromatic amines, anhydrides, and ketones were identified. A total of 116 compounds were tentatively identified, 22 for the first time, of which 16 possessed mutagenic activity. However, a lack of correlation between chemical composition and fraction mutagenicity in the medium polarity fractions, especially in the river sediment, was evidenced. The occurrence of multiple interactions between components in spiked organic extracts is demonstrated.

Bioassay-directed chemical characterization of genotoxic agents in the dissolved and particulate water phases of the Besos and Llobregat Rivers (Barcelona, Spain).

Particulate (greater than 0.22 microns) and dissolved phases of water concentrates (600 mL) of Llobregat and Besos Rivers (Barcelona, Spain), were tested in the Salmonella/microsome assay, tester strains TA98 and TA100. Most of them showed significant mutagenic activity. However, independently of the application of exogenous metabolic activation, the dimethylsulfoxide extracts of the particulate matter exhibited a stronger mutagenic activity than the dissolved phase. This indicated that both rivers are chronically polluted by frameshift and base-pair substitution mutagens and promutagens. In order to investigate their identity, a bioassay-directed column chromatography fractionation of the base-neutrals isolated from the dissolved and particulate phases of Besos river water (7 L) was carried out. The mutagenic activity (TA98) was higher in presence of S9 and was recovered in the more polar fractions, where several mutagenic agents were identified by capillary GC-MS in the negative ion chemical ionization mode (NICI). Among them, o-tolidine, nitroquinoline, nitroaniline, dichlorobenzidine and several aromatic quinones were candidates for fraction mutagenicity.

Transformation of Substituted Fluorenes and Fluorene Analogs by Pseudomonas sp. Strain F274.

Pseudomonas sp. strain F274, previously shown to catabolize fluorene via fluorenone and its angular dioxygenation, 2(prm1),3(prm1)-dihydroxy-2-carboxybiphenyl, phthalate, and protocatechuate, was examined for its ability to transform substituted fluorenes and S- and N-heterocyclic analogs. Halogen- and methyl-substituted fluorenes were metabolized to correspondingly substituted phthalates via attack on the unsubstituted ring. In the case of 1-methylfluorene, initial oxidation of the methyl group to carboxyl prevented all other transformations but 9-monooxygenation. This strain also oxidized the S-heteroatoms and benzylic methylenic groups of fluorene analogs. No angular dioxygenation of S- and N-heterocycles was observed.

Isolation and characterization of a fluorene-degrading bacterium: identification of ring oxidation and ring fission products.

An Arthrobacter sp. strain, F101, able to use fluorene as the sole source of carbon and energy, was isolated from sludge from an oil refinery wastewater treatment plant. During growth in the presence of fluorene, four major metabolites were detected and isolated by thin-layer chromatography and high-performance liquid chromatography. 9-Fluorenol, 9H-fluoren-9-one, and 3,4-dihydrocoumarin were identified by UV spectra, mass spectrometry, and 300-MHz proton nuclear magnetic resonance. The fourth metabolite has been characterized, but precise identification was not possible. Since strain F101 is not able to grow with fluorenone, two different pathways of fluorene biodegradation are suggested: one supports cell growth and produces 3,4-dihydrocoumarin as an intermediate and probably the unidentified metabolite, and the other produces 9-fluorenol and 9H-fluoren-9-one and appears to be a dead-end route.

Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons.

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.

Oxidation of naphthenoaromatic and methyl-substituted aromatic compounds by naphthalene 1,2-dioxygenase.

Oxidation of acenaphthene, acenaphthylene, and fluorene was examined with recombinant strain Pseudomonas aeruginosa PAO1(pRE695) expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone and cis- and trans-acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Nonspecific dehydrogenase activities present in the host strain led to the conversion of both of the acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was oxidized spontaneously to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. The action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in the formation of benzylic alcohols, i.e., methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially nonspecific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene.

New metabolites in the degradation of fluorene by Arthrobacter sp. strain F101.

Identification of new metabolites and demonstration of key enzyme activities support and extend the pathways previously reported for fluorene metabolism by Arthrobacter sp. strain F101. Washed-cell suspensions of strain F101 with fluorene accumulated 9-fluorenone, 4-hydroxy-9-fluorenone, 3-hydroxy-1-indanone, 1-indanone, 2-indanone, 3-(2-hydroxyphenyl) propionate, and a compound tentatively identified as a formyl indanone. Incubations with 2-indanone produced 3-isochromanone. The growth yield with fluorene as a sole source of carbon and energy corresponded to an assimilation of about 34% of fluorene carbon. About 7.4% was transformed into 9-fluorenol, 9-fluorenone, and 4-hydroxy-9-fluorenone. Crude extracts from fluorene-induced cells showed 3,4-dihydrocoumarin hydrolase and catechol 2,3-dioxygenase activities. These results and biodegradation experiments with the identified metabolites indicate that metabolism of fluorene by Arthrobacter sp. strain F101 proceeds through three independent pathways. Two productive routes are initiated by dioxygenation at positions 1,2 and 3,4, respectively. meta cleavage followed by an aldolase reaction and loss of C-1 yield the detected indanones. Subsequent biological Baeyer-Villiger reactions produce the aromatic lactones 3,4-dihydrocoumarin and 3-isochromanone. Enzymatic hydrolysis of the former gives 3-(2-hydroxyphenyl) propionate, which could be a substrate for a beta oxidation cycle, to give salicylate. Further oxidation of the latter via catechol and 2-hydroxymuconic semialdehyde connects with the central metabolism, allowing the utilization of all fluorene carbons. Identification of 4-hydroxy-9-fluorenone is consistent with an alternative pathway initiated by monooxygenation at C-9 to give 9-fluorenol and then 9-fluorenone. Although dioxygenation at 3,4 positions of the ketone apparently occurs, this reaction fails to furnish a subsequent productive oxidation of this compound.

Actions of a versatile fluorene-degrading bacterial isolate on polycyclic aromatic compounds.

Pseudomonas cepacia F297 grew with fluorene as a sole source of carbon and energy; its growth yield corresponded to an assimilation of about 40% of fluorene carbon. The accumulation of a ring meta-cleavage product during growth and the identification of 1-indanone in growth media and washed-cell suspensions suggest that strain F297 metabolizes fluorene by mechanisms analogous to those of naphthalene degradation. In addition to fluorene, strain F297 utilized for growth a wide variety of polycyclic aromatic compounds (PACs), including naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene, and dibenzothiophene. Fluorene-induced cells of the strain also transformed 2,6-dimethylnaphthalene, biphenyl, dibenzofuran, acenaphthene, and acenaphthylene. The identification of products formed from those substrates (by gas chromatography-mass spectrometry) in washed-cell suspensions indicates that P. cepacia F297 carries out the following reactions: (i) aromatic ring oxidation and cleavage, apparently using the pyruvate released for growth, (ii) methyl group oxidations, (iii) methylenic oxidations, and (iv) S oxidations of aromatic sulfur heterocycles. Strain F297 grew with a creosote-PAC mixture, producing an almost complete removal of all aromatic compounds containing 2 to 3 rings in 14 days, as demonstrated by gas chromatography analysis of the remaining PACs recovered from cultures. The identification of key chemicals confirmed that not only are certain compounds depleted but also the anticipated reaction products are found.

Isolation and characterization of (+)-1,1a-dihydroxy-1-hydrofluoren-9-one formed by angular dioxygenation in the bacterial catabolism of fluorene.

Transformation of fluorene by washed cells of fluorene-grown Pseudomonas sp. F274 yielded 1,la-dihydroxy-1-hydrofluoren-9-one (up to 100 mg/l) as the stable product of angular dioxygenation of 9-fluorenone. Structural identity of the angular keto-diol was established by 13C- and 1H-NMR, gas chromatography- and direct probe-mass spectrometry. Definitive assignment of 1,1a-dioxygenation, but not 4,4a-, was based on the isolation and rigorous identification of 1-hydroxyfluoren-9-one as the exclusive product of acidic dehydration. Chiral 1H-NMR analysis and optical rotation of isolated 1,1a-dihydroxy-1-hydrofluoren-9-one ([alpha]D = + 132.1 degrees) are indicative of a single enantiomer with an inferred cis-stereochemistry of the hydroxyl groups. This compound is evidently an intermediate of fluorene catabolism by this strain and not a dead-end product because its formation is transient in washed cell incubations and ultimately it is completely consumed with the formation of acidic metabolites.